Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes debilitating acute and chronic arthritis. Infection by CHIKV induces a robust immune response that is characterized by production of type I IFNs, recruitment of innate and adaptive immune cells, and development of neutralizing Abs. Despite this response, chronic arthritis can develop in some individuals, which may be due to a failure to eliminate viral RNA and Ag and/or persistent immune responses that cause chronic joint inflammation. In this review, based primarily on advances from recent studies in mice, we discuss the innate and adaptive immune factors that control CHIKV dissemination and clearance or contribute to pathogenesis.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne enveloped alphavirus in the Togaviridae family. CHIKV has a single-stranded positive sense RNA genome that encodes four nonstructural proteins (nsP1, nsP2, nsP3, and nsP4) and five structural proteins (capsid, E3, E2, 6K, and E1) from two open reading frames. CHIKV was first isolated in Tanzania in 1952 and has caused explosive outbreaks throughout Africa, India, Southeast Asia, and Polynesia (1, 2). CHIKV emerged in the Caribbean in 2013 and has spread throughout Central and South America, with autochthonous transmission reported in Florida (3). The outbreak in the Americas has resulted in more than 1.8 million suspected cases (4). Historically, CHIKV was transmitted principally by Aedes aegypti mosquitoes, but in 2006 the virus acquired a single mutation (A226V) in the E1 protein that facilitated enhanced replication and transmission in Aedes albopictus mosquitoes, which expanded its geographical range (5). There are three genotypes of CHIKV that are highly conserved, with 95.2–97% identity at the amino acid level: the East/Central/South African and Asian genotypes are more closely related than the more distantly related West African genotype (6, 7). Following a short incubation period after mosquito bite, CHIKV infection in humans can cause fever, rash, malaise, myalgia, and debilitating polyarthralgia and polyarthritis that usually lasts for 1–4 wk (8). Depending on the study, ~10–60% of affected individuals develop chronic arthritis that lasts for months to years following infection (912). CHIKV infection rarely results in mortality, although it has been reported in the elderly, infants, and immunocompromised (1315). Currently no approved vaccines or therapeutics are available to prevent CHIKV infection or treat disease at the acute or chronic stages.

Over the past decade, the immunobiology of CHIKV infection and disease has been studied intensively in laboratory animal models, primarily in mice but also in some nonhuman primate species. Experimental infection of different strains of immunocompetent mice (e.g., C57BL/6, CD1, IRC) results in an acute disease similar to that in humans, including high viremia, viral replication in joint and muscle tissues, synovitis, and myositis (1619). After inoculation of CHIKV into the skin, the virus likely replicates in fibroblasts, mesenchymal cells, and osteoblasts (20, 21). CHIKV induces a local cytokine and chemokine response that recruits NK cells, macrophages, inflammatory monocytes, and CD4+ and CD8+ T cells (16, 17, 20). Damage from viral replication and immune cell infiltration results in local edema, extensive myofiber degeneration, and injury and loss of mesenchymal cells lining the synovium and periosteum (22). Moreover, CHIKV infection of osteoblasts elevates the ratio of receptor activator of NF-κB ligand to osteoprotegerin in the ankle and knee, which increases osteoclast generation and can promote bone loss (23). In mice, CHIKV infection causes a biphasic pattern of swelling in the ipsilateral inoculated foot, with a small peak between 2 and 3 d postinfection (dpi) and a second, larger peak at 6–7 dpi (17, 24). The first peak is most likely due to extensive viral replication in the foot, which results in cell death, cytokine production, and tissue edema. The second peak occurs as infectious virus is cleared from the blood and within tissues, and is associated with the influx of inflammatory cells into joints of the foot and surrounding tissues, causing more edema, myositis, and synovitis. This histological observation suggests that the second, more prominent peak is driven by immune-mediated response and damage. Although infectious virus is cleared by 7 dpi, CHIKV RNA can be detected in joints (e.g., feet and ankles, and wrists) for ≥4 wk postinfection. Mice infected with a CHIKV strain encoding firefly luciferase showed bioluminescent signal in the foot at 45 dpi (25). Using mice lacking specific factors of innate and adaptive immunity, researchers have identified some of the key immune correlates of CHIKV disease pathogenesis and protection (Fig. 1, Table I) and have related them to observations from human cohort studies.

FIGURE 1.

Overview of CHIKV and immune-mediated pathogenesis in mice. CHIKV infection of the footpad results in edema and inflammation from viral infection, cell death, cytokine production, and immune cell infiltration. Foot swelling is biphasic, with the first (1) peak occurring 2–3 dpi followed by a second (2) peak at 6–7 dpi. (1) CHIKV infects fibroblasts (orange cells), mesenchymal cells, and osteoblasts. In this figure, infection is indicated with viral RNA present inside a cell with a plasma membrane colored orange. PRRs are triggered during cellular infection, resulting in activation of transcription factors, ultimately producing type I IFNs. Type I IFN and the ISG response are necessary to prevent severe disease. In addition, PRR and IFN signaling induces secretion of proinflammatory cytokines and chemokines, which recruit innate and adaptive immune cells to the site of infection, driving inflammation. Depletion of NK cells reduces foot swelling, suggesting a pathogenic role. Macrophages (MΦ) and inflammatory monocytes have dual protective and pathogenic roles in CHIKV arthritis. Depletion of macrophages reduces swelling but also can result in a neutrophil-mediated immunopathogenesis. Osteoblasts can be infected by CHIKV, which promotes osteoclastogenesis and bone reabsorption. γδ+ T cells prevent monocyte recruitment and joint inflammation. (2) CHIKV infection induces a neutralizing Ab (NAb) response that eliminates infectious virus from circulation and tissues. Effector CD4+ T cells are recruited to musculoskeletal tissues and secrete IFN-γ. Depletion of CD4+ T cells results in reduced joint swelling. Enhancement of the Foxp3+ CD4+ Treg response results in reduced joint swelling, cytokine production, and effector CD4+ T cell activation. Surprisingly, CD8+ T cells do not seem to function in acute pathogenesis or viral clearance, at least in mice. CHIKV RNA and Ag persist in joint tissues for extended periods and may serve as pathogen-associated molecular patterns for chronic immune activation and inflammation. Macrophages and monocytes are suggested to be a reservoir for chronic infection.

FIGURE 1.

Overview of CHIKV and immune-mediated pathogenesis in mice. CHIKV infection of the footpad results in edema and inflammation from viral infection, cell death, cytokine production, and immune cell infiltration. Foot swelling is biphasic, with the first (1) peak occurring 2–3 dpi followed by a second (2) peak at 6–7 dpi. (1) CHIKV infects fibroblasts (orange cells), mesenchymal cells, and osteoblasts. In this figure, infection is indicated with viral RNA present inside a cell with a plasma membrane colored orange. PRRs are triggered during cellular infection, resulting in activation of transcription factors, ultimately producing type I IFNs. Type I IFN and the ISG response are necessary to prevent severe disease. In addition, PRR and IFN signaling induces secretion of proinflammatory cytokines and chemokines, which recruit innate and adaptive immune cells to the site of infection, driving inflammation. Depletion of NK cells reduces foot swelling, suggesting a pathogenic role. Macrophages (MΦ) and inflammatory monocytes have dual protective and pathogenic roles in CHIKV arthritis. Depletion of macrophages reduces swelling but also can result in a neutrophil-mediated immunopathogenesis. Osteoblasts can be infected by CHIKV, which promotes osteoclastogenesis and bone reabsorption. γδ+ T cells prevent monocyte recruitment and joint inflammation. (2) CHIKV infection induces a neutralizing Ab (NAb) response that eliminates infectious virus from circulation and tissues. Effector CD4+ T cells are recruited to musculoskeletal tissues and secrete IFN-γ. Depletion of CD4+ T cells results in reduced joint swelling. Enhancement of the Foxp3+ CD4+ Treg response results in reduced joint swelling, cytokine production, and effector CD4+ T cell activation. Surprisingly, CD8+ T cells do not seem to function in acute pathogenesis or viral clearance, at least in mice. CHIKV RNA and Ag persist in joint tissues for extended periods and may serve as pathogen-associated molecular patterns for chronic immune activation and inflammation. Macrophages and monocytes are suggested to be a reservoir for chronic infection.

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Table I.
Clinical, immune, and viral phenotypes after CHIKV infection in mice
Immune Function and Mouse Strain (Age)aVirus Strain (Genotype)Clinical/Swelling/PathologyVirological and Immune CharacterizationRefs.
WT 
 Neonatal (9–12 d) 06.21 (ECSA); CHIKV-21 (ECSA) 40–60% Survival V: detectable in muscle, serum, brain, liver, lung (32, 34, 42
I: robust cytokine and IFN response by 24 h pi 
 Young (3–4 wk) SL15649 (ECSA); AF15561 (Asian) Inoculated foot swelling; edema, tenosynovitis, tendinitis V: detectable in foot, serum, muscle, liver, brain; persistent RNA in joints and spleen (16, 18, 63
I: innate and adaptive cell infiltrations; IgM and IgG produced 
 Adult (6–12 wk) Asian isolate; LR2006-OPY1 (ECSA) Inoculated foot swelling; edema, arthritis, tenosynovitis (ECSA > Asian) V: detectable in serum, foot, muscle, spleen, LN, liver; persistent RNA in joints (17, 80
I: proinflammatory cytokine induction; innate and adaptive cell infiltration; IgG2c dominant 
Antiviral pathways 
Tlr3−/− (young) SGPO11 (ECSA) ↑ Foot swelling V: ↑ viremia and tissue titers (30
I: ↑ infiltration of macrophages, neutrophils; ↓ CD4+ T cells; ↓ IgG neutralization 
Tlr3−/− CHIKV-21 ND V: no Δ in titers (1–3 dpi) (20
Trif−/− (adult) LR2006-OPY1 ↑ Foot swelling V: ↑ viremia (31
I: ↓ type I IFNs 
Rig-I−/− (ICR/129/C57BL/6) CHIKV-21 ND V: no Δ in tissue, slight increase in viremia (20
Mda5−/− (ICR/129/C57BL/6) CHIKV-21 ND V: no Δ in viremia or tissues (72 h pi) (20
Mavs−/− (adult) CHIKV-21; LR2006-OPY1 ↑ Foot swelling V: ↑ viremia (20, 31
I: no type I IFNs 
Myd88−/− (adult) CHIKV-21; LR2006-OPY1 No Δ in foot swelling V: ↑ viremia and tissue titers (20, 31
I: ↓ type I IFNs 
Irf3−/− Irf7−/− (adult) LR2006-OPY1; CHIKV-21 ↑ Foot swelling; 100% lethal V: ↑ tissue burden (31, 32
I: ↑ IL-6, TNF-α, IFN-γ; no type I IFNs; shock syndrome 
Irf3−/− (adult) LR2006-OPY1; CHIKV-21 ↑ Foot swelling V: no Δ in foot titer or viremia (31, 32
I: no Δ type I IFNs or no type I IFNs 
Irf7−/− (adult) LR2006-OPY1; CHIKV-21 ↑ Foot swelling V: ↑ viremia; no Δ in foot titer I; no type I IFNs (31, 32
Stat1−/− (129/Sv) CHIKV-21, LR2006-OPY1, 37997 (WA) ↑ Foot swelling in ips. and contra. (LR); 100% lethal V: ↑ tissue titers (20, 35
Ifnar1−/− (C57BL6, 129/Sv) (adult) CHIKV-21 100% Lethal V: ↑ viremia and tissue titers (32, 34, 35
Rsad2−/− (young) SGP 011 (Asian) ↑ Foot swelling V:↑ viremia late; ↑ foot titer (38
I: ↑ F4/80+ macrophages 
Gadd34−/− (neonatal) CHIKV-21 100% lethal V: ↑ tissue titers (36
I: ↓ type I IFNs 
Ifitm3−/− (young) LR2006-OPY1 ↑ Foot swelling V: ↑ tissue titers (39
I: ↑ macrophage infection 
Bst2−/− 181/25 (Asian) No swelling (attenuated virus) V: ↑ viremia and tissue titers (40
I: ↓ type I IFNs and IFN-γ 
Isg15−/− (neonatal) 06.21 100% Lethal V: no Δ (42
I: ↑ cytokines; shock syndrome 
Innate cellular responses 
 Macrophage depletion (clodronate) (adult) LR2006-OPY1 ↓ Foot swelling V: ↑ viremia (late) (17
Ccr2−/− (adult) LR2006-OPY1 ↑ Foot swelling V: no Δ (53
I: ↑ neutrophils and eosinophils; ↑ inflammatory cytokines 
 NK cell depletion (young) LR2006-OPY1; CNR20235 (Asian) ↓ Foot swelling early; no Δ (Asian) V: ND (59
 γδTCR−/− (young) SL15649 ↑ Foot swelling; ↓ weight gain V: no Δ in viral burden or clearance (61
I: ↑ inflammatory and regulatory monocytes; ↑ cytokines 
Adaptive cellular responses 
Rag1−/− or Rag2−/− (adult; young) LR2006-OPY1; SL15649, SGP11, AF15561 ↓ Foot swelling; chronic synovitis, tendinitis V: ↑ viremia (acute) and persistent virus in periphery and serum (18, 25, 62, 63
I: ↓ cellular infiltrates; ↑ in neutrophils (chronic) 
Cd8−/− or CD8 depletion (adult) SGP11; LR2006-OPY1-Fluc No Δ V: no Δ (25
Cd4−/− (adult) SGP11 ↓ Foot swelling V: no Δ in viremia (25, 80
I: ↓ cellular infiltration in foot; ↓ Ab response 
 CD4+ T cell depletion (adult) SGP11; LR2006-OPY1-Fluc ↓ Foot swelling V: no Δ in viremia (25
I: ↓ recruitment of CD8+ T cells; no Δ in macrophages or neutrophils 
MHCII−/− (adult) LR2006-OPY1 ↓ Foot swelling V: no Δ in viremia (62
I: no IgG1 produced; ↓ IgG2c; ↓ cellular infiltrates 
 Treg expansion (IL-2/anti-IL-2 complex) (young) SGP011 ↓ Foot swelling V: no Δ viremia (66
I: ↓ proinflammatory cytokines; ↓ effector CD4+ T cells and proliferation 
Ifng−/− (adult) SGP11 Mild ↑ foot swelling V: ↑ viremia (25
μMT−/− (adult) LR2006-OPY1; SGP11 ↑ And prolonged foot swelling V: ↑ and chronic viremia (62, 80
I: no Δ in cellular infiltrates 
Immune Function and Mouse Strain (Age)aVirus Strain (Genotype)Clinical/Swelling/PathologyVirological and Immune CharacterizationRefs.
WT 
 Neonatal (9–12 d) 06.21 (ECSA); CHIKV-21 (ECSA) 40–60% Survival V: detectable in muscle, serum, brain, liver, lung (32, 34, 42
I: robust cytokine and IFN response by 24 h pi 
 Young (3–4 wk) SL15649 (ECSA); AF15561 (Asian) Inoculated foot swelling; edema, tenosynovitis, tendinitis V: detectable in foot, serum, muscle, liver, brain; persistent RNA in joints and spleen (16, 18, 63
I: innate and adaptive cell infiltrations; IgM and IgG produced 
 Adult (6–12 wk) Asian isolate; LR2006-OPY1 (ECSA) Inoculated foot swelling; edema, arthritis, tenosynovitis (ECSA > Asian) V: detectable in serum, foot, muscle, spleen, LN, liver; persistent RNA in joints (17, 80
I: proinflammatory cytokine induction; innate and adaptive cell infiltration; IgG2c dominant 
Antiviral pathways 
Tlr3−/− (young) SGPO11 (ECSA) ↑ Foot swelling V: ↑ viremia and tissue titers (30
I: ↑ infiltration of macrophages, neutrophils; ↓ CD4+ T cells; ↓ IgG neutralization 
Tlr3−/− CHIKV-21 ND V: no Δ in titers (1–3 dpi) (20
Trif−/− (adult) LR2006-OPY1 ↑ Foot swelling V: ↑ viremia (31
I: ↓ type I IFNs 
Rig-I−/− (ICR/129/C57BL/6) CHIKV-21 ND V: no Δ in tissue, slight increase in viremia (20
Mda5−/− (ICR/129/C57BL/6) CHIKV-21 ND V: no Δ in viremia or tissues (72 h pi) (20
Mavs−/− (adult) CHIKV-21; LR2006-OPY1 ↑ Foot swelling V: ↑ viremia (20, 31
I: no type I IFNs 
Myd88−/− (adult) CHIKV-21; LR2006-OPY1 No Δ in foot swelling V: ↑ viremia and tissue titers (20, 31
I: ↓ type I IFNs 
Irf3−/− Irf7−/− (adult) LR2006-OPY1; CHIKV-21 ↑ Foot swelling; 100% lethal V: ↑ tissue burden (31, 32
I: ↑ IL-6, TNF-α, IFN-γ; no type I IFNs; shock syndrome 
Irf3−/− (adult) LR2006-OPY1; CHIKV-21 ↑ Foot swelling V: no Δ in foot titer or viremia (31, 32
I: no Δ type I IFNs or no type I IFNs 
Irf7−/− (adult) LR2006-OPY1; CHIKV-21 ↑ Foot swelling V: ↑ viremia; no Δ in foot titer I; no type I IFNs (31, 32
Stat1−/− (129/Sv) CHIKV-21, LR2006-OPY1, 37997 (WA) ↑ Foot swelling in ips. and contra. (LR); 100% lethal V: ↑ tissue titers (20, 35
Ifnar1−/− (C57BL6, 129/Sv) (adult) CHIKV-21 100% Lethal V: ↑ viremia and tissue titers (32, 34, 35
Rsad2−/− (young) SGP 011 (Asian) ↑ Foot swelling V:↑ viremia late; ↑ foot titer (38
I: ↑ F4/80+ macrophages 
Gadd34−/− (neonatal) CHIKV-21 100% lethal V: ↑ tissue titers (36
I: ↓ type I IFNs 
Ifitm3−/− (young) LR2006-OPY1 ↑ Foot swelling V: ↑ tissue titers (39
I: ↑ macrophage infection 
Bst2−/− 181/25 (Asian) No swelling (attenuated virus) V: ↑ viremia and tissue titers (40
I: ↓ type I IFNs and IFN-γ 
Isg15−/− (neonatal) 06.21 100% Lethal V: no Δ (42
I: ↑ cytokines; shock syndrome 
Innate cellular responses 
 Macrophage depletion (clodronate) (adult) LR2006-OPY1 ↓ Foot swelling V: ↑ viremia (late) (17
Ccr2−/− (adult) LR2006-OPY1 ↑ Foot swelling V: no Δ (53
I: ↑ neutrophils and eosinophils; ↑ inflammatory cytokines 
 NK cell depletion (young) LR2006-OPY1; CNR20235 (Asian) ↓ Foot swelling early; no Δ (Asian) V: ND (59
 γδTCR−/− (young) SL15649 ↑ Foot swelling; ↓ weight gain V: no Δ in viral burden or clearance (61
I: ↑ inflammatory and regulatory monocytes; ↑ cytokines 
Adaptive cellular responses 
Rag1−/− or Rag2−/− (adult; young) LR2006-OPY1; SL15649, SGP11, AF15561 ↓ Foot swelling; chronic synovitis, tendinitis V: ↑ viremia (acute) and persistent virus in periphery and serum (18, 25, 62, 63
I: ↓ cellular infiltrates; ↑ in neutrophils (chronic) 
Cd8−/− or CD8 depletion (adult) SGP11; LR2006-OPY1-Fluc No Δ V: no Δ (25
Cd4−/− (adult) SGP11 ↓ Foot swelling V: no Δ in viremia (25, 80
I: ↓ cellular infiltration in foot; ↓ Ab response 
 CD4+ T cell depletion (adult) SGP11; LR2006-OPY1-Fluc ↓ Foot swelling V: no Δ in viremia (25
I: ↓ recruitment of CD8+ T cells; no Δ in macrophages or neutrophils 
MHCII−/− (adult) LR2006-OPY1 ↓ Foot swelling V: no Δ in viremia (62
I: no IgG1 produced; ↓ IgG2c; ↓ cellular infiltrates 
 Treg expansion (IL-2/anti-IL-2 complex) (young) SGP011 ↓ Foot swelling V: no Δ viremia (66
I: ↓ proinflammatory cytokines; ↓ effector CD4+ T cells and proliferation 
Ifng−/− (adult) SGP11 Mild ↑ foot swelling V: ↑ viremia (25
μMT−/− (adult) LR2006-OPY1; SGP11 ↑ And prolonged foot swelling V: ↑ and chronic viremia (62, 80
I: no Δ in cellular infiltrates 
a

All mice are on the C57BL/6 background unless otherwise noted. The age of mice at time of infection is listed after genotype; if not listed, the age was not reported.

↑, increased; ↓, decreased; Δ, change; contra., contralateral; ECSA, East Central South African; I, immune; ips., ipsilateral; LN, lymph node; LR, La Reunion Island; ND, no data; pi, postinfection; V, virological.

Recognition of CHIKV RNA and induction of antiviral pathways.

The mammalian host response to CHIKV infection starts locally in the skin at the site of inoculation and also occurs in the underlying muscle and joint tissues (18, 26). The CHIKV RNA genome can trigger host pattern recognition receptors (PRRs), including endosomal TLRs (TLR3 and TLR7) and cytoplasmic RIG-I–like (RIG-I and MDA5) receptors, which activate downstream adaptor molecules (e.g., TRIF, MyD88, and MAVS) to induce nuclear translocation of IRF3- and type I IFN–dependent antiviral responses (2729). Although this scheme generally is accepted for many viruses, reports conflict regarding the necessity of individual PRRs in the antiviral response against CHIKV in mice. For example, one group showed a protective effect of TLR3, as Tlr3−/− mice sustained increased viremia and tissue viral burden with augmented foot swelling and edema, infiltration of CD11b+ F4/80+ macrophages and CD11b+ Ly6G+ neutrophils, and reduced numbers of CD4+ T cells (30). Depletion of the remaining CD4+ T cells in the Tlr3−/− mice diminished foot swelling but also reduced the number of infiltrating neutrophils; the authors suggested that the increased recruitment of neutrophils in the CHIKV-infected Tlr3−/− mice promotes the observed enhanced musculoskeletal inflammation (30). In this study, TLR3 stimulation in hematopoietic cells controlled CHIKV viremia, whereas TLR3 signaling in nonhematopoietic cells resulted in reduced clinical disease. Remarkably, a second group observed no virological differences between Tlr3−/− and wild-type (WT) mice (20). Potential reasons for the disparity in viral infection phenotype may be related to the age of mice inoculated and the method of virus detection. The first group used 3-wk-old mice and analyzed viral burden by quantitative RT-PCR (30). The second group did not disclose the age of mice used and determined viral burden by 50% tissue culture infective dose analysis (20). An antiviral role for TLR3 is supported by studies with Trif−/− mice, which showed increased foot swelling and viremia after CHIKV inoculation (31).

The RIG-I–like receptors also restrict CHIKV infection and pathogenesis, as a deficiency of MAVS resulted in enhanced viral replication and foot swelling; however, animals deficient in either RIG-I or MDA5 did not show these phenotypes, suggesting a redundant role for these two PRRs in the sensing of CHIKV RNA (20, 31). Additional studies need to be performed with mice lacking MAVS, RIG-I, and MDA5 to evaluate for effects on the composition and activation state of immune cell infiltrates. Although no direct CHIKV infection studies have been reported in Tlr7−/− mice, Myd88−/− mice supported increased viremia and dissemination with relatively minor differences in foot swelling, compared with WT mice (20, 31). Because MyD88 is an adaptor molecule downstream of multiple TLRs and other signaling receptors (e.g., IL-1R), direct infection and analysis of immune responses of other TLR-deficient mice are required.

PRR signaling promotes nuclear translocation of IFN regulatory (IRF1, IRF3, IRF5, and IRF7) and NF-κB transcription factors, which induce expression of type I IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines and chemokines (28). In the absence of IRF7, no bioactive type I IFNs were produced in the serum of CHIKV-infected animals (31, 32). There are conflicting reports regarding the role of IRF3 in the induction of type I IFNs after CHIKV infection. One group showed no type I IFNs produced in Irf3−/− mice, whereas a second reported equivalent levels of type I IFNs compared with levels in WT mice (31, 32). Variations in the results could be related to different CHIKV strains and route of inoculation. However, CHIKV infection of Irf3−/−Irf7−/− double knockout (DKO) mice resulted in a lethal shock syndrome characterized by massive production of proinflammatory cytokines (IFN-γ, CCL2, IL-6, and TNF-α), thrombocytopenia, and high tissue viral burden (31). The DKO mice also had severe edema, hemorrhaging in the foot, vasculitis, and exudative arthritis that resulted in pronounced swelling (31). In comparison, increased foot swelling was detected in Irf3−/− or Irf7−/− single knockout mice after CHIKV infection, but only the Irf7−/− mice showed increased viremia (31). Consistent with a protective effect of type I IFN signaling, mice lacking the type I IFN receptor (Ifnar1−/− C57BL/6 or 129/Sv animals) or downstream transcription factor STAT1 also succumbed to CHIKV infection, although the time to death was longer in Irf3−/−Irf7−/− DKO compared with Ifnar1−/− mice (20, 3235). This finding suggests that additional transcription factors (e.g., IRF1 and IRF5) may contribute to the type I IFN response after CHIKV infection. Bone marrow reconstitution studies in WT, Irf3−/−Irf7−/− DKO, and Ifnar1−/− mice showed that whereas type I IFNs can be produced by both hematopoietic and nonhematopoietic cells after CHIKV infection, IFN signaling in nonhematopoietic cells controls viral replication (20, 32). This result correlates with the preferential targeting of nonhematopoietic cells, such as fibroblasts, muscle cells, and osteoblasts, by CHIKV (20, 21).

A limited set of ISGs have been described as inhibitors of CHIKV infection in vivo. Protein kinase R (PKR) and OAS3 are ISGs activated during CHIKV replication that lead to translation inhibition, apoptosis, and degradation of single-stranded RNA (27, 36, 37). GADD34 and other proteins are upregulated following PKR activation and promote type I IFN and IL-6 production. GADD34-deficient cells sustained increased CHIKV infection and minimal IFN-β production, which recapitulates the phenotype observed in Eif2ak1 (gene encoding PKR)–deficient cells (36). Gadd34−/− neonatal mice succumbed to CHIKV infection more rapidly with reduced IFN-β production and increased viral burden in tissues (36). Induction of RSAD2, which encodes the protein viperin, was observed in CHIKV-infected CD14+ human monocytes (38). Rsad2−/− fibroblasts supported increased CHIKV replication, and Rsad2−/− mice sustained increased viremia, foot edema, and inflammation, which were associated with infiltration of F4/80+ macrophages (38). As the endoplasmic targeting domain of Rsad2 is required to inhibit CHIKV infection in vitro, viperin likely is recruited to the endoplasmic reticulum, where it induces a stress response that activates PKR and blocks CHIKV translation (38). A recent study reported that IFITM3 inhibits CHIKV infection by blocking viral fusion in cells (39). Ifitm3−/− mice sustained increased tissue viral burden, which was mediated in part by greater infection of CD11b+ F4/80lo and CD11blo F4/80hi macrophages (39). Another ISG, Bst2 (gene encoding tetherin), blocks CHIKV from budding from the cell. Bst2−/− mice had enhanced viremia with reduced expression of type I and II IFNs (40); the antiviral effect of Bst2 occurs even though CHIKV nsp1 suppresses expression of Bst2 mRNA and protein (41). ISG15 modulates CHIKV pathogenesis in neonatal mice. Isg15−/− mice have increased lethality without changes in viral burden (42). In the absence of Isg15, an uncontrolled cytokine response occurs, with markedly increased production of TNF-α, IL-1β, and IL-6, resulting in a shock syndrome, similar to that observed in Ifnar1−/− neonatal mice (42). These results correlate with findings from human infants infected with CHIKV, who have higher serum cytokine levels (e.g., type I IFNs, IL-1Rα, CXCL10, and IL-12p40/70) than do adults (42). Beyond these few studies that have established antiviral functions of individual ISGs against CHIKV in vivo, other ISGs (e.g., IRF1, C6orf150, HSPE, P2RY6, SLC15A3, and SLC25A28) have been suggested to have anti-CHIKV activity based on ectopic expression in cells (43). These ISGs require further study to confirm their inhibitory activity in vivo and determine their mechanism of action.

Human cohort studies have characterized the proinflammatory cytokine and chemokine response during acute CHIKV infection. One group showed increased levels of IL-2R, IL-5, IL-6, IL-7, IL-10, IL-15, IFN-α, CXCL9, CXCL10, human growth factor, fibroblast growth factor–basic, and vascular endothelial growth factor and decreased levels of eotaxin, epidermal growth factor, and IL-8 with CHIKV infection compared with healthy controls (44). Individuals with severe CHIKV disease had increased levels of IL-1β and IL-6 and decreased levels of RANTES compared with individuals with nonsevere cases (44). A different study correlated increased levels of IL-6, IL-12, IL-15, CXCL10, CCL2, IFN-α, and IL-1Rα with higher viremia (45), and a separate study identified increased levels of CXCL10 and CXCL9 in the serum of patients with mild-to-severe disease compared with nonsymptomatic patients (46). Individuals with persistent arthralgia had increased levels of IL-6 and GM-CSF (45). Collectively, these studies suggest that specific cytokines (e.g., IL-6) may be linked to CHIKV disease severity and pathogenesis.

Innate cellular immune responses.

Soon after acute CHIKV infection, monocyte-derived macrophages migrate to the site of viral replication. This occurs in part because the macrophage chemoattractant CCL2 (MCP-1) is induced during CHIKV infection (17, 47), presumably by fibroblasts, monocytes, and endothelial and epithelial cells (48). Infiltrating and tissue-resident macrophages produce IL-6, TNF-α, and GM-CSF, and may act as a reservoir for chronic CHIKV RNA and Ag (4951). As clodronate treatment resulted in reduced foot swelling and increased viremia at late time points, macrophages appear to promote clinical disease while aiding in clearance of infectious virus (17). Consistent with this observation, blockade of macrophage recruitment using the small molecule bindarit, which modulates NF-κB signaling and CCL2 production (52), reduced clinical disease and inflammation in synovial and skeletal muscle tissues (47). However, Ccr2−/− mice showed an opposing phenotype, with enhanced foot swelling and inflammation with cartilage erosion (53). In this genetic knockout model, the lack of macrophage chemotaxis resulted in compensatory infiltration of neutrophils and eosinophils into the foot at early and late times postinfection, respectively, and changes to the cytokine milieu (53).

By the second peak of foot swelling, F4/80+ CD11b+ macrophages increase expression of markers of M2 differentiation, including arginase 1 and Ym1, which may aid in tissue repair but also could contribute to incomplete clearance of CHIKV, leading to persistence of viral RNA (54). Whereas some macrophage recruitment is required to prevent an unbalanced immunological cellular composition and protect against pathological inflammation, excessive recruitment increases edema and inflammatory cytokines. Inflammatory monocytes (CD11b+ F4/80lo) also traffic to musculoskeletal tissues during infection in mice (17) and could serve as a secondary reservoir. Circulating CHIKV-infected CD14+ monocytes can be detected during acute illness in humans, and the percentage of infected monocytes correlates with viremia (55).

Although NK cells accumulate in musculoskeletal tissues shortly after CHIKV infection (16, 17), their protective or pathological function remains uncertain. In one study evaluating NK (CD3 CD56+) and NKT-like (CD3+ CD56+) cells during acute and convalescent CHIKV infection in humans, NK cells were elevated during both phases and NKT-like cells were increased only during the chronic phase. During acute infection, a higher fraction of NK cells produced perforin with a trend toward enhanced cytotoxic activity, compared with those from convalescent samples (56). Another study reported that NK cells during the acute stage have increased expression of CD69 and HLA-DR activation markers (57). These cells also had increased expression of NKp44, CD57, ILT2, CD8α, and NKG2C and decreased expression of NKp30, NKp46, NKG2A, and CD161, suggesting that a unique subset of NK cells may be activated after CHIKV infection (57). Another cohort of individuals in the late-acute to early-chronic stage of disease showed an increase in the percentage of NK cells (CD3 CD123 CD14 CD11c CD19 CD45+ CD38+ CD16+) from isolated PBMCs (58). In mice, a recent study compared the immune responses postinfection of strains of the ECSA and Asian genotypes; mice infected with an ECSA virus had increased foot edema and a higher number of NK cells compared with animals infected with an Asian virus. Depletion of NK cells reduced foot swelling in animals infected with the ECSA, but not Asian, strain of CHIKV (59). These results suggest that NK cell–dependent immune-mediated pathogenesis may vary among CHIKV genotypes.

Because CHIKV is inoculated into the skin by mosquitoes, γδ T cells have been speculated to have antiviral and/or immunomodulatory roles (60). Indeed, CHIKV infection increases the number of LCA+ CD3+ γδ T cells in the ipsilateral foot and draining lymph node. TCRγδ−/− mice have increased foot swelling during the second peak, myositis with loss of myocytes, enhanced numbers of inflammatory (LCA+ CD11b+ CD11c Ly6C+) and regulatory (LCA+ CD11b+ CD11c Ly6C) monocytes, and elevated levels of proinflammatory cytokines with no change in viral burden (61). Thus, γδ T cells appear to have a protective regulatory role of immune pathology primarily through their ability to modulate inflammatory cell composition in infected tissues.

The adaptive immune response to CHIKV infection has a dual role in protection and pathogenesis. Although it is absolutely required for the clearance of infectious virus and protection against subsequent infection, it also contributes to the pathological changes observed during the acute phase (Fig. 1). Mice deficient in both B and T cells (Rag1−/− or Rag2−/−) had higher levels of viremia during the acute phase, and infectious virus was sustained in circulation and peripheral tissues for ≥3 mo, which resulted in chronic arthritis and muscle inflammation (18, 25, 62, 63). Although the absence of an adaptive immune response results in a failure to clear circulating infectious virus, these mice developed less peak foot swelling, with reduced myositis and synovitis, implicating a role for T and/or B cells in the early stages of immune pathogenesis (18, 25).

T cell response.

In humans, CHIKV infection results in the activation of CD8+ T cells (as judged by increased expression of CD69 and HLA-DR), with peak levels in peripheral blood detected soon after symptom onset (64). Individuals 7–10 wk postinfection still have a high percentage of activated CD38+ HLA-DR+ CD8+ T cells in circulation, compared with healthy patients (58). In mice, CD8+ T cells are recruited to affected musculoskeletal tissue within the first week of infection (25). Surprisingly, genetic or acquired deficiencies of CD8+ T cells did not affect foot swelling, joint inflammation, or viremia, compared with controls, suggesting that CD8+ T cells do not contribute to disease or clearance of infectious virus during the acute phase (25). Many questions remain as to the functions (or lack thereof) of CD8+ T cells during acute and possibly chronic CHIKV infection, especially in regard to viral clearance and persistence. Possible explanations for their apparent lack of antiviral activity include lack of polyfunctionality, cellular exhaustion, or viral antagonism of priming.

Large numbers of CD4+ T cells migrate into the joint capsule (synovium) within the first week of CHIKV infection, and these cells produce high levels of IFN-γ (25). Unlike that seen with CD8+ T cells, genetic or acquired deficiencies of CD4+ T cells (25) or MHC class II molecules (62) resulted in reduced foot swelling and decreased recruitment of CD8+ T cells without a significant effect on viremia (25). This phenotype is not due to lack of IFN-γ production because Ifng−/− mice show increased viremia at late time points and a minimally increased foot swelling compared with WT controls after CHIKV infection (25). Depletion of CD4+ T cells with Abs did not affect the numbers or kinetics of infiltrating CD45+ CD11b+ Ly6G monocytes/macrophages or CD45+ CD11b+ Ly6G+ neutrophils into the foot, suggesting that acute clinical disease in the joint and adjacent muscle tissues is due preferentially to CD4+ T cells (25). The role of CD4+ T cells during chronic disease has not yet been elucidated in mice. A study examining synovial biopsies from humans with chronic CHIKV disease showed a high fraction of activated CD69+ CD4+ T cells, suggesting these cells may contribute to chronic disease, although the mechanism remains unknown (65).

Although relatively small numbers of Foxp3+ CD4+ regulatory T cells (Tregs) migrate to the joint space after CHIKV infection, they appear to modify disease (66). Treg expansion after administration of an IL-2/anti–IL-2 complex reduced foot swelling, tissue edema, and cytokine and chemokine mRNA expression, including IL-6, IFN-γ, CXCL10, and IL-10. Higher numbers of Tregs correlated with reduced numbers of IFN-γ+-producing CD4+ T cells in the foot and decreased proliferation of effector CD4+ T cells in the draining lymph node, presumably by downregulating expression of costimulatory molecules on APCs (66). Thus, pharmacological strategies to augment Treg expansion could limit the pathological CD4+ T cell response and minimize immune-mediated disease. However, the impact of such interventions on viral persistence and chronic disease warrants further study.

B cell response.

Treatment of CHIKV-infected Rag1−/− mice with exogenous neutralizing mAbs eliminates infectious virus from circulation, but as the Ab levels wane infectious virus re-emerges, highlighting the importance of Abs in controlling, but not completely clearing, infection (18, 62, 63). Indeed, passive transfer of purified monoclonal or polyclonal anti-CHIKV Abs can protect immunocompromised mice from lethal disease when administered prior to or shortly postinfection (24, 6772).

Many mouse and human anti-CHIKV mAbs neutralize CHIKV in vitro and protect in vivo against acute or chronic musculoskeletal disease in mice and nonhuman primates (7275). Neutralizing Abs target the envelope glycoproteins, which are displayed on the virion surface as trimers of E2/E1 heterodimers (76). Most strongly neutralizing Abs target the A and B domains on the E2 protein, and Abs against the B domain of CHIKV broadly inhibit infection against several arthritogenic alphaviruses (74). Neutralizing anti-CHIKV Abs can inhibit at multiple steps of the virus life cycle, including blockade of virus attachment, entry, pH-dependent fusion in the endosome, new virion assembly, release of the virion from the plasma membrane, and cell-to-cell spread (24, 68, 72, 74, 7779). Apart from this, Abs may protect against CHIKV infection through their effector functions, including Ab-mediated cellular cytotoxicity, complement activation, and virus opsonization.

In mice, anti-CHIKV IgM can be detected within a few days of infection and begins to wane during the second week (80). This coincides with the development of an IgG response, which increases through the acute phase and remains high during the chronic phase (80). High titers of neutralizing Ab are detected by the end of the second week of infection (80). In μMT mice lacking mature B cells, viremia is increased, compared with WT mice, and maintained essentially for the life of the animal; the enhanced infection of μMT mice also is associated with increased and prolonged foot swelling, compared with WT mice (62, 80). In CHIKV-infected humans, virus-specific IgM can be detected for months, whereas isotype switching to IgG occurs within 1 wk of infection (65, 81). One study using cohorts of CHIKV-infected patients from acute and chronic phases of disease found that the IgG response was dominated by the IgG3 isotype (82). Individuals with an early IgG3 response paradoxically had higher levels of viremia and more severe acute clinical disease, but developed persistent arthralgia less often (82). The IgG3 neutralizing Abs reportedly are directed at a single, linear peptide epitope (E2EP3; STKDNFNVYKATRPYLAH) that is located at the N terminus of the E2 protein (83, 84). CHIKV-infected nonhuman primates generated anti-E2EP3 Abs, and mice vaccinated with the E2EP3 peptide had reduced viremia and foot swelling upon challenge (83).

Studies in mice have identified factors that modify the protective Ab response and control of CHIKV infection. In MHCII−/− mice, class switching from IgM to IgG occurs in the absence of T cell help; although an IgG2c response is generated, the levels are reduced compared with those in WT mice (62). CHIKV-infected Tlr3−/− mice showed increased IgG titers with reduced neutralization capacity, indicating a qualitative defect of Ab function in the absence of signaling by this PRR (30). A recent study identified a mechanism of CHIKV RNA persistence in peripheral tissues through evasion of neutralizing Ab (63). An attenuated, nonpersistent strain of CHIKV was neutralized more efficiently than a pathogenic, persistent strain by mouse or human CHIKV immune serum. A single amino acid (residue 82) in the E2 protein of the pathogenic strain affected neutralization by Abs targeting the E2 B domain and allowed for evasion (63).

Although acute CHIKV infection generates a robust immune response that eliminates circulating infectious virus, questions remain regarding the immunobiology of chronic disease. Synovial biopsies from chronically infected humans showed CHIKV-infected perivascular macrophages, large numbers of CD14+ macrophages/monocytes, and activated CD56+ CD69+ NK cells (65). CHIKV-infected rhesus macaques or cynomolgus macaques also show persistence of CHIKV RNA in their spleens, muscle, and joint tissue and CHIKV Ag in CD68+ macrophages of lymphoid organs (49, 85). These results suggest that the chronic phase of disease may be sustained by immune activation from persistent viral RNA and Ag, which results in continuous production of inflammatory cytokines and chemokines in synovial and muscle tissue. It remains unclear whether CHIKV RNA persists because of active viral replication or whether an ineffective immune response fails to eliminate infected cells. Regardless of the mechanism, persistent arthralgia and arthritis occur in a significant fraction of affected individuals. Because chronic CHIKV disease mimics seronegative rheumatoid arthritis (58, 86), drugs approved to treat rheumatoid arthritis have been reported anecdotally as possible treatments for chronic CHIKV arthritis. Retrospective studies in humans have described the use of nonsteroidal anti-inflammatory drugs, methotrexate, and corticosteroids to treat chronic CHIKV arthritis, with limited success (86, 87). As we generate a more sophisticated understanding of the interplay between immunity and chronic CHIKV disease, the development or repurposing of immune targeted therapies may be a realistic intervention in the near future.

We thank Jonathan Miner for critical review of the manuscript.

This work was supported by National Institutes of Health Grants R01 AI073755, R01 AI104972, and R01 AI114816. J.M.F. was supported by National Institutes of Health Grant T32 AI007172.

Abbreviations used in this article:

CHIKV

Chikungunya virus

DKO

double knockout

dpi

day postinfection

ISG

IFN-stimulated gene

PKR

protein kinase R

PRR

pattern recognition receptor

Treg

regulatory T cell

WT

wild-type.

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M.S.D. is a consultant for Inbios, Sanofi, and Takeda Pharmaceuticals and is on the Scientific Advisory Boards of Moderna and OraGene. The other author has no financial conflicts of interest.